A biologically active substance uniformly dispersed microsphere and a sustained release formulation comprising the same

ABSTRACT

The present application provides a microsphere comprising a lactic acid-glycolic acid copolymer (PLGA) or polylactide (PLA) as a main component, in which a biologically active substance is uniformly dispersed, wherein an average volume-based particle diameter of the microsphere is 1 μm or more and 150 μm or less, and a variation coefficient of area ratios in six regions is 0.35 or less, wherein the area ratios in six regions are calculated by (s/A)×100 (%) wherein the six regions are prepared by preparing a cross section observation sample obtained by cutting the microsphere; observing the cross section observation sample with an electron microscope at a magnification capable of confirming the biologically active substance in the microsphere or a higher magnification; and dividing the electron microscope observation image into six regions; and A is an area of a respective divided region, and s is a sum of cross section areas of the biologically active substance included in the respective divided region. The microsphere of the present invention can appropriately control the initial release amount of the biologically active substance and its release rate during a subsequent release period, and can continuously release the biologically active substance in vivo for a predetermined period of time.

TECHNICAL FIELD

The present inventions relate to a microsphere in which a biologicallyactive substance is uniformly dispersed and a sustained releaseformulation comprising the same. The present inventions specificallyrelate to a microsphere comprising a lactic acid-glycolic acid copolymer(PLGA) or polylactide (PLA) as a main component in which a biologicallyactive substance is uniformly dispersed and a sustained releaseformulation comprising the same.

BACKGROUND ART

Recently, a microsphere or nanosphere has attracted attention as asustained release formulation of a medicine containing a biologicallyactive substance or the like. A microsphere generally refers to aformulation having a particle diameter of 1 μm to about 150 μm, and aformulation smaller than that having a particle diameter less than 1 μmis referred to as a nanosphere. For example, when a biologically activesubstance is incorporated in a biodegradable synthetic or naturalpolymer, the biologically active substance can be continuously releasedlocally, or the biologically active substance can be targeted to atissue.

A sustained release microsphere formulation which gradually releases abiologically active substance at a constant rate, needs to be, forexample, a formulation in which a biodegradable polymer, a biologicallyactive substance, an additive, a solvent and the like are appropriatelycontrolled. In order for a sustained release microsphere formulation toeffectively exhibit a pharmacological effect in vivo for a predeterminedperiod of time, it is necessary to continuously release the biologicallyactive substance in vivo for a predetermined period of time, byappropriately controlling the initial release amount of the biologicallyactive substance and its release rate during a subsequent releaseperiod.

One of the important factors for determining the release rate of thebiologically active substance is a kind of biodegradable polymers.Particularly, the most widely used lactic acid-glycolic acid copolymer(polylactide-co-glycolide acid, PLGA) and polylactide (PLA) being apolymer of lactic acid has a different biodegradation rate depending onits physicochemical properties such as a ratio of lactic acid andglycolic acid, its molecular weight, its affinity with water and thelike, and thus, the biodegradation rate can be adjusted to a desiredrelease period (Patent Literature 1).

Furthermore, in addition to that, a particle diameter of the microsphereand a dispersion state of the biologically active substance in themicrosphere are related, in order to suppress an abnormal initialrelease amount (an initial burst) of a biologically active substance andto control its release rate during a release period to be constant. Inspite of a problem of yield, the particle diameter of the microspherecan be adjusted to a desired particle diameter by an operation such asfiltration. However, the dispersion state of the biologically activesubstance in the microsphere has been reported only as uniform, and hasnot been confirmed.

A microsphere of PLGA or PLA can be produced using, for example, amethod of drying in liquid, a spray drying method, a spray freeze dryingmethod, a drying method using a supercritical fluid process, a doubleemulsification method, or the like. When a biologically active substanceis lipophilic, the most common production method among these methods isa method of drying in liquid in which PLGA or PLA and the biologicallyactive substance are dissolved or dispersed in an organic solvent, mixedand emulsified with an aqueous solution in which polyvinyl alcohol (PVA)is dissolved, and the solvent is removed from the emulsion.

Patent Literature 1 discloses a method of producing a sustained releasemicrosphere containing a biodegradable polymer such as PLGA and apeptide medicine by a spray drying method, a spray freeze drying methodor a drying method using a supercritical fluid process. However, PatentLiterature 1 does not describe how much the particle diameter of thesustained release microsphere varies, whether or not the peptidemedicine is uniformly dispersed in the sustained release themicrosphere, and whether a uniformly dispersed microsphere is obtained.

Patent Literature 2 discloses a method of producing PLGA microparticlesby a method of drying in liquid using a mixed solvent comprising ahalogenated hydrocarbon and a water-immiscible organic solvent having asolubility of a medicine of 0.3% (WN) or more. Patent Literature 2describes that particle diameters (median diameters) of themicroparticles obtained in Production Examples 1 and 2 are respectively14 and 16 μm, but does not describe the dispersion state of the medicinein the microparticles.

Patent Literature 3 discloses PLGA nanoparticles containing a medicine.These nanoparticles are mainly intended for targeting to a specifictissue, and are nanoparticles of several tens nm to several hundreds nmwhich can pass through microscopic pores of blood capillaries. However,Patent Literature 3 does not describe a microsphere of 1 μm or more muchlarger than these nanoparticles. Even with the technique of PatentLiterature 3, a person skilled in the art could not produce amicrosphere having a particle diameter of 1 μm or more.

Patent Literature 4 discloses a formulation which releases leuprorelinacetate of a luteinizing hormone releasing hormone derivative, duringfrom about 1 month to several months by subcutaneous injection. Theformulation has a problem that a distribution of the particle diametersis very wide from 1 μm to 400 μm. Therefore, Patent Literature 5proposes as a method for solving this problem, a method of producing amicrosphere in which a biologically active substance is encapsulated ina polymer for a carrier by a double emulsification method. However,Patent Literature 5 does not describe the dispersion state of themedicine in the leuprorelin acetate containing the microspheres obtainedin Examples 1 to 5.

Patent Literature 6 discloses a microsphere which reduces a chronic painfor at least 28 days (672 hours). The microsphere comprises abiodegradable polymer and a local anesthetic (a biologically activesubstance), and releases about 75% of the local anesthetic during about72 hours, and about 80 to 90% of the local anesthetic during about 120hours. This suggests that the distribution of the local anesthetic inthe microsphere is not uniform in the microsphere, and is biased in theouter side. From the SEM (scanning electron microscope) image of a crosssection of the microsphere described in FIG. 2, the dispersion state ofthe local anesthetic cannot be confirmed.

Patent Literature 7 discloses a core shell structure microsphere inwhich a core contains solid aripiprazole, and the surface of the core iscoated with a shell containing a biodegradable polymer. As describedabove, the microsphere of Patent Literature 7 is not a microsphere inwhich the biologically active substance is uniformly dispersed. Further,in the electron microscope photograph of a cross section obtained bycutting the microsphere obtained in the example shown in FIG. 5, thedispersion state of aripiprazole cannot be confirmed in the shell.

CITATION LIST Patent Literature

-   Patent Literature 1: JP 2005-035994-   Patent Literature 2: JP 2005-015476-   Patent Literature 3: JP 4856752-   Patent Literature 4: JP 2653255-   Patent Literature 5: JP 2014-224114-   Patent Literature 6: JP 2016-069378-   Patent Literature 7: JP 2010-531303

SUMMARY OF THE INVENTION Technical Problem

A biodegradable polymer microsphere having an average volume-basedparticle diameter of 1 μm or more and 150 μm or less cannot be put inpractice as its release period is designed, unless the distribution of abiologically active substance (hereinafter, may be referred to as amedicine) in the microsphere is controlled. For example, when thebiologically active substance is biased near the surface of themicrosphere, a large amount of the biologically active substance isreleased from the microsphere at the initial period after administrationto generate the problem of the initial burst. On the other hand, whenthe biologically active substance is biased in the center of themicrosphere or when the microsphere is in a state of a core shellstructure, the biologically active substance cannot be releasedcontinuously from the initial period. Therefore, a state in which thebiologically active substance is uniformly dispersed in themicroparticle, is desirable. When it is in a dispersed state in whichlarge masses of the biologically active substance are scattered, thebiologically active substance cannot be released continuously from theinitial period. Similarly, when empty holes are not controlled, asimilar problem occurs in the release of the biologically activesubstance.

When pharmacokinetics are actually investigated using a small animalsuch as a rat, many variations in the release rate and release profilesometimes occur. The reason is often concluded as an individualdifference of rats. However, if the dispersion state of a biologicallyactive substance in the microsphere is uniform, most of the variationswill be more improved, and decomposition rate will be controlled by akind and molecular weight of PLGA, and release of the biologicallyactive substance from the microsphere can be realized as designed.

Uniform dispersion of the biologically active substance in themicrosphere is the absolute condition for continuously releasing thebiologically active substance in vivo for a predetermined period oftime. However, the uniform dispersion has not been searched at present.Since the particle diameter of the microsphere is large unlike that ofthe nanoparticle, homogenization of the microsphere is generallydifficult. Therefore, it is necessary to confirm the dispersion state ofthe biologically active substance in the microsphere. For that, a crosssection observation sample obtained by cutting a microsphere particle isprepared, and is observed with an electron microscope at a magnificationcapable of confirming the biologically active substance in themicrosphere or a higher magnification, so that the dispersion state canbe confirmed. This can be easily performed, and is certain.

FIG. 1 is a microphotograph of the microcapsule sustained releaseformulation leuplin (registered trademark) for injection 1.88 mg (TakedaPharmaceutical Company Limited), which corresponds to the sustainedrelease microcapsule of a LH-RH derivative described in PatentLiterature 4.

The formulation includes various sizes of particles from large particlesto small particles. FIG. 2 is an SEM (scanning electron microscope)image of a cross section of the particle of about 6 μm which wasselected as a representative particle. It is understood by confirmationof an edge effect in an image, or by an EDS (energy dispersive X-rayspectrometer), that large dispersion bodies indicated by arrows in FIG.2 are empty holes. FIG. 3 is an image prepared by dividing the SEM crosssection image of FIG. 2 into six regions (Region 1 to Region 6); anaveraging process in the pixel range of 3×3 using a commercial imageanalysis software iTEM (TEM camera control, image analysis software,EMSIS GmbH); contrast optimization by a process of highlighting the edgepart; a binarization process; a process of removing noises andhighlighting particles with low contrast by image processing; a secondaveraging process in the pixel range of 3×3; and a process ofhighlighting the edge part. Based on FIG. 3, a variation coefficient ofthe area ratios: (s/A)×100(%), wherein A is an area of a respectivedivided region, and s is a sum of cross section areas of thebiologically active substance included in the respective divided region,was calculated, and the variation coefficient was 1.06. By performing inthis way, the dispersion state of the biologically active substance canbe confirmed from the cross section. Since the variation coefficientexceeds 0.35, and the biologically active substance is not uniformlydispersed in the particle, the formulation cannot appropriately controlthe release rate during the release period.

Accordingly, an object of the present invention is to provide amicrosphere capable of appropriately controlling the initial releaseamount of a biologically active substance and its release rate during asubsequent release period, and continuously releasing the biologicallyactive substance in vivo for a predetermined period of time.

Solution to the Problem

The present inventors earnestly studied to solve the above problem. As aresult of that, the present inventors have found that by a microspherewherein a variation coefficient of the area ratios of a biologicallyactive substance in a respective region prepared by dividing an electronmicroscope cross section observation image of the microsphere into sixregions, is 0.35 or less, the biologically active substance is uniformlydispersed in the microsphere, empty holes are not present, and themicrosphere can appropriately control the initial release amount of abiologically active substance and its release rate during a subsequentrelease period, and can continuously release the biologically activesubstance in vivo for a predetermined period of time. Thus, the presentinventors have accomplished the present inventions. Namely, the presentinventions are as follows.

[1] The first embodiment of the present invention is a microspherecomprising a lactic acid-glycolic acid copolymer (PLGA) or polylactide(PLA) as a main component, in which a biologically active substance isuniformly dispersed,

wherein an average volume-based particle diameter of the microsphere is1 μm to 150 μm, and

a variation coefficient of area ratios in six regions is 0.35 or less,wherein the area ratios in six regions are calculated by (s/A)×100(%)wherein the six regions are prepared by preparing a cross sectionobservation sample obtained by cutting the microsphere; observing thecross section observation sample with an electron microscope at amagnification capable of confirming the biologically active substance inthe microsphere or a higher magnification; and dividing the electronmicroscope observation image into six regions; and A is an area of arespective divided region, and s is a sum of cross section areas of thebiologically active substance included in the respective divided region.

[2] The second embodiment of the present invention is the microsphereaccording to [1], wherein the biologically active substance is alipophilic biologically active substance.

[3] The third embodiment of the present invention is the microsphereaccording to [1] or [2], wherein an average volume-based particlediameter of the dispersed biologically active substance is 5 nm to 500nm.

[4] The fourth embodiment of the present invention is the microsphereaccording to any one of [1] to [3], wherein a content of thebiologically active substance in the microsphere is 0.35 to 1.5% bymass.

[5] The fifth embodiment of the present invention is a sustained releaseformulation comprising the microsphere according to any one of [1] to[4].

Advantageous Effects of the Invention

The microsphere of the present invention can appropriately control theinitial release amount of a biologically active substance and itsrelease rate during a subsequent release period, and can continuouslyrelease the biologically active substance in vivo for a predeterminedperiod of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SEM (scanning electron microscope) image of leuplin(registered trademark) for injection 1.88 mg (Takeda PharmaceuticalCompany Limited).

FIG. 2 shows an SEM image of a cross section of a representativeparticle of leuplin (registered trademark) for injection 1.88 mg (TakedaPharmaceutical Company Limited).

FIG. 3 shows an image prepared by dividing the cross section image ofFIG. 2 into six regions and a binarization process, for calculating arearatios: (s/A)×100(%), wherein A is an area of a respective dividedregion, and s is a sum of cross section areas of the biologically activesubstance included in the respective divided region.

FIG. 4 shows an SEM image of a cross section of the microsphere withouta biologically active substance of Reference Example 1.

FIG. 5 shows examples of a method of dividing a microsphere. (A) showsan example of dividing it into six regions concentrically. (B) shows anexample of dividing it into six regions longitudinally. (C) shows anexample of dividing it into six regions latitudinally.

FIG. 6-1 shows an SEM image of a cross section of the microsphere ofExample 1.

FIG. 6-2 shows an image prepared by dividing the cross section image ofFIG. 6-1 into six regions and a binarization process, for calculatingarea ratios: (s/A)×100(%), wherein A is an area of a respective dividedregion, and s is a sum of cross section areas of the biologically activesubstance included in the respective divided region.

FIG. 7-1 shows an SEM image of a cross section of the microsphere ofExample 3.

FIG. 7-2 shows an image prepared by enlarging the cross section image ofFIG. 7-1 and a binarization process.

FIG. 8-1 shows an SEM image of a cross section of the microsphere ofExample 4.

FIG. 8-2 shows an image prepared by dividing the cross section image ofFIG. 8-1 into six regions and a binarization process, for calculatingarea ratios: (s/A)×100(%), wherein A is an area of a respective dividedregion, and s is a sum of cross section areas of the biologically activesubstance included in the respective divided region.

FIG. 9 shows an image prepared by dividing the cross section image ofReference Example 1 into six regions and a binarization process, forcalculating area ratios: (s/A)×100(%), wherein A is an area of arespective divided region, and s is a sum of cross section areas of thebiologically active substance included in the respective divided region.

FIG. 10 shows an SEM image of a cross section of the microsphere ofComparative Example 2.

FIG. 11 shows an SEM image of a cross section of the microsphere ofComparative Example 3.

FIG. 12 shows an SEM image of a cross section of the microsphere ofComparative Example 4.

DESCRIPTION OF THE INVENTION

1. Microsphere

The microsphere of the present invention is a microsphere comprising alactic acid-glycolic acid copolymer (PLGA) or polylactide (PLA) as amain component, in which a biologically active substance is uniformlydispersed, wherein an average volume-based particle diameter of themicrosphere is 1 μm to 150 μm, and a variation coefficient of arearatios in six regions is 0.35 or less, wherein the area ratios in sixregions are calculated by (s/A)×100(%) wherein the six regions areprepared by preparing a cross section observation sample obtained bycutting the microsphere; observing the cross section observation samplewith an electron microscope at a magnification capable of confirming thebiologically active substance in the microsphere or a highermagnification; and dividing the electron microscope observation imageinto six regions; and A is an area of a respective divided region, and sis a sum of cross section areas of the biologically active substanceincluded in the respective divided region.

When a biologically active substance is biased in the surface layer orin the center of the microsphere, or when coarse particles, aggregatesor large empty holes, etc. are present in the microsphere, the abovevariation coefficient of area ratios becomes large. When the abovevariation coefficient of area ratios is 0.35 or less, a biologicallyactive substance is uniformly dispersed. In the microsphere of thepresent invention, the variation coefficient of area ratios ofoccupation of the biologically active substance relative to the area ofthe respective region, in the respective region obtained by dividing across section of the microsphere into six regions, is 0.35 or less,preferably 0.25 or less, more preferably 0.20 or less.

The microsphere of the present invention can appropriately control theinitial release amount of the biologically active substance and itsrelease rate during a subsequent release period, and can continuouslyrelease the biologically active substance in vivo for a predeterminedperiod of time.

<Observation of Cross Section of Microsphere>

A method of confirming a dispersion state of a biologically activesubstance in the microsphere is explained below.

The method can be performed by observing a cross section of themicrosphere with an electron microscope at a magnification capable ofclearly confirming the dispersed microparticles of the biologicallyactive substance. The electron microscope includes a transmissionelectron microscope (TEM) using transmitted electrons as an informationsource, a scanning electron microscope (SEM) detecting secondaryelectrons (backscattered electrons), etc. The electron microscope may beselected according to the sample to be observed. The fine structure ofnanospheres can be more observed with a transmission electronmicroscope. Specifically, the microsphere is coated with a thin film ofgold, platinum, platinum/palladium alloy, etc. In Examples, themicrospheres were coated with osmium. Then, the microsphere is firstfrozen with liquid nitrogen. After frozen, a cross section of FIB(focused ion beam) is prepared. That is, a cross section observationsample of the microsphere is prepared by irradiating a focused ion beamonto a sample using an FIB apparatus, and cutting out a structure at adesired position inside the sample. A preferable particle diameter ofthe microparticles of the biologically active substance dispersed in thebase material of PLGA or PLA is several tens nm to several hundreds nm,but the particle diameter may be several μm in some cases. An entirecross section of the microsphere is observed at an observationmagnification of an electron microscope capable of confirming thedispersed microparticles of the above preferable particle diameter.Usually, an observation magnification of an electron microscope is from2,500 to several hundreds of thousands or more. In addition, in the casewhere a higher magnification at which the entire microsphere cannot beobserved is used, the observed portions may be joined to observe theentire microsphere.

When the cross section image is divided into six regions, for example,as shown in FIGS. 5A to 5C, the cross section image may be divided intosix regions concentrically, or may be divided into six regions in thevertical or horizontal direction, or may be divided into six regionslatitudinally from the center. It is preferable to take a dividingmethod that remarkably shows a segregation state of the biologicallyactive substance. For example, when dividing into six regionsconcentrically (FIG. 5A), it is essential to divide concentrically bydividing the radius into six equal parts from the center point of themaximum diameter of the cross section of the microsphere. When dividinginto six regions in the vertical or horizontal direction (FIG. 5B), itis essential to divide into six regions at equal intervals. It isessential to divide into six regions in either direction parallel orperpendicular to the above maximum diameter. When dividing into sixregions latitudinally from the center (FIG. 5C), it is essential todivide into six regions latitudinally every 60° around the center pointof the above maximum diameter as a center. Depending on the constituentelements of the biologically active substance, an elementary analysismay be performed by analyzing the cross section of the microsphere usingan EDS (Energy Dispersive X-ray Spectrometer), and it can be alsoconfirmed whether or not it is an empty hole. In the case of emptyholes, it should not be integrated into the area of the biologicallyactive substance. When the EDS detection elements are not contained, thecross section observation sample may be stained with rutheniumtetroxide, osmium tetroxide, phosphotungstic acid, uranyl acetate,iodine or the like. It is also possible to identify the biologicallyactive substance by comparison with a microsphere without thebiologically active substance. The staining method is effective when itis difficult to obtain contrast in the electron microscope cross sectionobservation image. The above are explained just as examples, and thesample may be embedded with a resin, or a microtome may be used toprepare a cross section of the microsphere.

A method of calculating a cross section area is not particularlylimited, but it is preferable to use a commercial image analysissoftware. As a commercial image analysis software, various kind ofsoftware such as Image-Pro Plus (Media Cybernetics, Inc.), iTEM (TEMcamera control, image analysis software, EMSIS GmbH), etc. can be used.

<PLGA or PLA>

PLGA is a lactic acid-glycolic acid copolymer having a constitutionalunit derived from lactic acid and a constitutional unit derived fromglycolic acid. PLA is a polymer of lactic acid. PLGA may compriseanother biodegradable polymer such as polylactide (PLA), polyglycolide(PGA) and the like. The PLGA described herein is described as anexample, and the present invention is not limited to the described PLGA.

The molar ratio (L:G) of the constitutional unit (L) derived from lacticacid and the constitutional unit (G) derived from glycolic acid in PLGAis not particularly limited, and may be appropriately selected accordingto the intended purpose. A preferable molar ratio (L:G) is 1:99 to 99:1,more preferably 25:75 to 99:1, further preferably 30:70 to 90:10,particularly preferably 50:50 to 85:15. Only PLA may be used. Selectionof this molar ratio is important for realizing a uniform dispersionstate of a biologically active substance. Selection of a molecularweight of PLGA or PLA is also similarly important.

PLGA used in the microsphere of the present invention can be produced,for example, by heating and condensation polymerization of lactic acidand glycolic acid under a weakly reduced pressure using an ion exchangeresin as a catalyst. In this case, lactide may be used in place oflactic acid. PLGA may be a commercially available product. Acommercially available product may be purchased from, for example,FUJIFILM Wako Pure Chemical Corporation, Taki Chemical Co., Ltd., EvonicRohm GmbH, Merck KGaA, Sigma-Aldrich Co., LLC, and the like.

The content of PLGA or PLA in the microsphere of the present inventionis not particularly limited, and may be appropriately selected accordingto the intended purpose. The content is preferably 1% by mass or more,more preferably 30% by mass or more and 95% by mass or less, andparticularly preferably 50% by mass or more and 90% by mass or less.

<Microsphere>

The microsphere of the present invention includes PLGA or PLA and abiologically active substance. The microsphere may further contain adispersing agent and another component, if necessary. The biologicallyactive substance, a dispersing agent, another component and the like aredispersed in the base material of PLGA or PLA in the microsphere.

[Biologically Active Substance]

A biologically active substance contained in the microsphere of thepresent invention is not particularly limited, and may be appropriatelyselected according to the intended purpose. The biologically activesubstance may be, for example, a pharmaceutical compound, a functionalfood compound, a functional cosmetic compound, and the like. Amicrosphere containing a pharmaceutical compound can be suitably used,for example, as a sustained release pharmaceutical formulation. Thebiologically active substance includes both a lipophilic biologicallyactive substance and a hydrophilic biologically active substance. Apreferable biologically active substance includes a lipophilicbiologically active substance. A lipophilic biologically activesubstance means, for example, a substance having a log P value ofwater/octanol distribution coefficient of 3 or more, and a biologicallyactive substance not contained in a lipophilic biologically activesubstance is classified as a hydrophilic biologically active substance.The water/octanol distribution coefficient can be measured according tothe Japanese Industrial Standard: JIS Z 7260-107 (2000): Flask shakingmethod. The biologically active substance is not particularly limited aslong as a sustained release formulation comprising the biologicallyactive substance is desired, and may be appropriately selected accordingto the intended purpose. The biologically active substance includes anyform of a salt, hydrate, and the like.

The biologically active substance is uniformly dispersed in themicrosphere of the present invention. By adopting such constitution, themicrosphere can appropriately control the initial release amount of thebiologically active substance and its release rate during a subsequentrelease period, and can continuously release the biologically activesubstance in vivo for a predetermined period of time. Uniform dispersionof the biologically active substance in the microsphere can becontrolled by the content of the biologically active substance, relativeto the total amount of the microsphere. A preferable content of thebiologically active substance varies depending on the biologicallyactive substance, and is, for example, 0.1 to 3% by mass, preferably 0.3to 2% by mass, more preferably 0.35 to 1.5% by mass, further morepreferably 0.5 to 1.25% by mass, relative to the total amount of themicrosphere.

The average volume-based particle diameter of the dispersedmicroparticles of the biologically active substance is preferably 5 nmto 500 nm, more preferably 10 nm to 400 nm, and further preferably 20 nmto 200 nm.

[Dispersing Agent]

A dispersing agent may be used for dispersing the biologically activesubstance. The dispersing agent may be a low molecular weight dispersingagent or a high molecular weight polymer dispersing agent. A lowmolecular weight dispersing agent means a compound having a mass averagemolecular weight less than 15,000. A high molecular weight polymerdispersing agent means a compound having a mass average molecular weightof 15,000 or more, including repeated covalent bonds between one or moreof monomers.

The low molecular weight dispersing agent is not particularly limited aslong as it is acceptable for a pharmaceutical compound, a functionalfood compound, a functional cosmetic compound, and the like, and may beappropriately selected according to the intended purpose. A specificexample thereof includes a lipid, a saccharide, a cyclodextrin, an aminoacid, an organic acid, another component, and the like. These may beused alone or in combination of two kinds or more thereof.

The lipid is not particularly limited, and may be appropriately selectedaccording to the intended purpose. The lipid may be, for example, amedium chain or long chain monoglyceride, diglyceride or triglyceride, aphospholipid, a vegetable oil (e.g. soybean oil, avocado oil, squaleneoil, sesame oil, olive oil, corn oil, rape-seed oil, safflower oil,sunflower oil, etc.), a fish oil, a flavoring oil, a water-insolublevitamin, a fatty acid, and a mixture thereof, a derivative thereof andthe like. These may be used alone or in combination of two kinds or morethereof.

The sugar is not particularly limited, and may be appropriately selectedaccording to the intended purpose. The sugar includes, for example,glucose, mannose, idose, galactose, fucose, ribose, xylose, lactose,sucrose, maltose, trehalose, turanose, raffinose, maltotriose, acarbose,water-soluble cellulose, synthetic cellulose, sugar alcohol, glycerin,sorbitol, lactitol, maltitol, mannitol, xylitol, erythritol, polyol, anda derivative thereof, and the like. These may be used alone or incombination of two kinds or more thereof.

The another component is not particularly limited and may beappropriately selected according to the intended purpose. The anothercomponent is preferably one which can be used for a medicine so far.

<Average Volume-Based Particle Diameter>

The average volume-based particle diameter of the microsphere of thepresent invention is 1 μm or more and 150 μm or less, preferably 10 μmor more and 100 μm or less, and more preferably 20 μm or more and 75 μmor less. The average volume-based particle diameter may be measuredusing a laser diffraction particle size distribution measuringapparatus. In the present invention, when the average volume-basedparticle diameter exceeds 150 μm, the problem of an initial burst occursdue to non-uniform dispersibility of the biologically active substancein the microsphere, which tends to cause aggregation and sedimentation,and to lead to difficult processing in a subsequent step. When theaverage volume-based particle diameter is smaller than 1 μm, the problemof an initial burst occurs remarkably.

The microsphere of the present invention can appropriately control theinitial release amount of a biologically active substance and itsrelease rate during a subsequent release period, and can continuouslyrelease the biologically active substance in vivo for a predeterminedperiod of time.

2. Sustained Release Formulation

By using the microsphere of the present invention, a sustained releaseformulation containing the microsphere can be prepared. The sustainedrelease formulation of the present invention can appropriately controlthe initial release amount of a biologically active substance and itsrelease rate during a subsequent release period, and can continuouslyrelease the biologically active substance in vivo for a predeterminedperiod of time, and a pharmacological effect can be effectivelyexhibited.

The sustained release formulation of the present invention can be simplyadministered as an injection, an implant or a transdermal formulation,directly to a lesion such as a muscle, subcutaneous tissue, bloodvessel, organ, joint cavity, tumor, or the like. It may be administeredas various other formulations. For example, when preparing asustained-release preparation of the present invention as an injectionformulation, the sustained release injection formulation as an aqueoussuspension may be prepared together with a dispersing agent (Tween 80,HCO 60, carboxy methylcellulose, sodium alginate, etc.), a preservative(methylparaben, propylparaben, etc.), an isotonizing agent (sodiumchloride, mannitol, sorbitol, glucose, etc.) and the like.Alternatively, the sustained release injection formulation as an oilsuspension may be prepared with a vegetable oil such as soybean oil,sesame oil, corn oil, and the like.

3. Method of Producing Microsphere

<Step of Producing Microsphere>

The method of producing the microsphere of the present inventionincludes at least the step of forming particles, and may further includethe step of filtration and sterilization, the step of removing a goodsolvent, and other steps, if necessary.

<Step of Forming Particles>

In the step of forming particles, it is preferable to use a pulverizingapparatus in which pulverization is performed between a plurality ofprocessing surfaces being capable of approaching to and separating fromeach other, at least one of which rotates relative to the other, whichis described in JP 2009-132871 or JP 2011-189348. The step of formingparticles is performed, for example, by continuously feeding to thepulverizing apparatus a solution of PLGA or PLA and a biologicallyactive substance obtained by dissolving or dispersing PLGA or PLA andthe biologically active substance in a good solvent of PLGA or PLA, anda solution containing a poor solvent of PLGA or PLA to prepareemulsified particles; and removing the good solvent from the producedparticles to precipitate the microsphere of the present invention. Here,“dispersing” includes dispersing the biologically active substance as asolid in a good solvent of PLGA or PLA; emulsifying the biologicallyactive substance in a good solvent of PLGA or PLA; forming a w/oemulsion containing an aqueous solution of a hydrophilic biologicallyactive substance and a good solvent of PLGA or PLA; and the like.

The solution of PLGA or PLA and a biologically active substance is notparticularly limited as long as it is a solution in which PLGA or PLAand the biologically active substance are dissolved or dispersed in agood solvent of PLGA or PLA, and may be appropriately selected accordingto the intended purpose. The good solvent is not particularly limited,and may be appropriately selected according to the intended purpose. Thegood solvent includes, for example, a halogenated aliphatic hydrocarbon,an aliphatic ester, an alcohol, a ketone, an ether, acetonitrile, andthe like. An example of the halogenated aliphatic hydrocarbon includesdichloromethane, chloroform, carbon tetrachloride, chloroethane,2,2,2-trichloroethane, and the like. An example of the aliphatic esterincludes ethyl acetate, propyl acetate, butyl acetate, and the like. Anexample of the alcohol includes an alcohol having low solubility inwater such as benzyl alcohol, phenyl alcohol, n-butanol, and the like.An example of the ketone includes a ketone having 3 to 6 carbon atoms(e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone,cyclohexanone, etc.), and the like. An example of the ether includes anether having 2 to 6 carbon atoms (e.g., dimethyl ether, methyl ethylether, diethyl ether, etc.), and the like. It is preferable to select asolvent having low solubility in water, from the view point of thecontent of the biologically active substance and for the purpose ofpreventing an initial burst. A good solvent is preferably a halogenatedaliphatic hydrocarbon, a ketone, and a mixture thereof, more preferablydichloromethane, acetone and a mixture thereof. These may be used aloneor in combination of two kinds or more thereof. The particle diametercan be controlled by changing a kind of the solvent or a mixing amountof the solvent.

A good solvent means a solvent having high solubility of PLGA or PLA,and a poor solvent means a solvent having low or no solubility of PLGAor PLA. A good solvent and a poor solvent are selected so that thebiologically active substance is not biased in each microsphere, and acoarse particle or an aggregate of particles is not generated. Inaddition, a good solvent and a poor solvent can be defined by, forexample, a quantity of PLGA or PLA which can be dissolved in 100 g ofthe solvent at 25° C. In the present invention, the good solvent ispreferably a solvent which dissolves 0.1 g or more, more preferably 0.2g or more, and still more preferably 0.5 g or more of PLGA or PLA. Thepoor solvent is preferably a solvent which dissolves only 0.05 g orless, more preferably 0.02 g or less, and still more preferably 0.01 gor less of PLGA or PLA. The poor solvent is not particularly limited,and may be appropriately selected according to the intended purpose, andwater is preferable.

A content of PLGA or PLA in a solution of PLGA or PLA and a biologicallyactive substance may be changed depending on the good solvent, dependingon the particle diameter of the intended microsphere, so that thebiologically active substance is uniformly dispersed in the microsphere.The content of PLGA or PLA is, for example, 1 to 30% by mass, preferably3 to 20% by mass, and more preferably 5 to 15% by mass. The content ofthe biologically active substance in the solution of PLGA or PLA may beappropriately changed according to the intended purpose, thepharmacological effect and the like, so that the biologically activesubstance is uniformly dispersed in the microsphere.

A stabilizer may be added to the poor solvent for further ensuringstability of the produced microsphere. The stabilizer is notparticularly limited, and may be appropriately selected according to theintended purpose. The stabilizer includes, for example, polyvinylalcohol (PVA), polyvinyl pyrrolidone (PVP), carboxy methylcellulose(CMC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose(HPMC), lecithin, Polysorbate 80, and the like, and polyvinyl alcohol(PVA) is preferable. Further, the concentration of the added stabilizeris preferably 0.01 to 20% by mass, more preferably 5% by mass or less.The preferable poor solvent is, for example, an aqueous solution of PVA,and the like.

The solution of PLGA of PLA and a biologically active substance and thesolution of a poor solvent is desirably prepared using a preparationapparatus such as a rotatory dispersing apparatus which realizes uniformmixing by applying a shearing force to a fluid, for example, by rotatinga stirring bar of various shapes such as a rod, a plate and a propellerin a tank, or by equipping with a screen rotating relative to a stirringbar. A stirring apparatus disclosed in JP 5147091 may be applied as apreferable example of the rotatory dispersing apparatus. It is necessaryto thoroughly mix the solution of PLGA or PLA and the poor solvent, foruniformly dispersing the biologically active substance in themicrosphere. For complete mixing, it is necessary to aim athomogenization at least on a molecular level. Incomplete mixing causesun-uniform dispersion state.

The rotatory dispersing apparatus may be a batch type one or acontinuous type one. When performed by a continuous type rotatorydispersing apparatus, a stirring energy can be appropriately controlled,by using an apparatus to continuously supply and discharge a fluid toand from the stirring tank, or using a continuous mixer without using astirring tank, or using a known stirring apparatus or a stirring means.Incidentally, the stirring energy is described in detail in JPH04-114725 by the present applicant. The stirring method in the presentinvention is not particularly limited, but may be performed using avarious shearing type, friction type, high-pressure jet type, ultrasonictype, etc, of a stirrer, a dissolver, an emulsifier, a disperser, ahomogenizer, or the like. An example thereof includes a continuous typeemulsifier such as ULTRA-TURRAX (IKA-Werke GmbH & Co. KG), POLYTRON(Kinematica AG), TK HOMOMIXER (Primix Corporation), Ebara Milder (EbaraCorporation), TK HOMOMETIC LINE FLOW (Primix Corporation), Colloid Mill(Kobelko Eco-Solutions, Co., Ltd.), Slasher (NIPPON COKE & ENGINEERING,Co., Ltd.), Trigonal Wet Pulverizer (Mitsui Miike Chemical EngineeringMachinery, Co., Ltd.), Cavitron (Euro Tech, Co., Ltd.), Fine Flow Mill(Pacific Machinery & Engineering, Co., Ltd.), and the like; a batch typeor continuous dual type emulsifier such as Clearmix (M. Technique Co.,Ltd.), Clearmix Dissolver (M. Technique Co., Ltd.), and the like.Further, it is desirable to use a stirring apparatus equipped with astirring blade rotating at high speed and equipped with a screen outsideof the stirring blade which discharges a fluid as a jet stream from anopening of the screen, particularly, the above Clearmix (M. TechniqueCo., Ltd.) and Clearmix Dissolver (M. Technique Co., Ltd.).

In the above pulverizing apparatus, it is possible to control theparticle diameter and the particle diameter distribution ofmicroparticles of PLGA or PLA by adjusting the contact pressure of therotating processing surfaces at a standstill period. As a result ofexperiments by the present inventors, the contact pressure is preferably20 g/cm² to 250 g/cm². When the contact pressure is lower than 20 g/cm²,the thin film is not stable and the particle diameter distributionbecomes wide. When the contact pressure is higher than 250 g/cm², it hasbeen found difficult to adjust the intended particle diameter. Thecontact pressure may be preferably 50 g/cm² to 200 g/cm², and morepreferably 80 g/cm² to 150 g/cm².

It is preferable to prevent coalescence of the respective microspheresformed by contacting the solution of PLGA or PLA and the biologicallyactive substance with the solution containing the poor solvent. As amethod of preventing the coalescence, the solution containing a poorsolvent is preferably added in a tank for recovering a solutiondischarged fluid beforehand, and is slowly stirred. By stirring, thecoalescence of the microspheres can be further suppressed. A rotatorydispersing apparatus is preferable for stirring, and Clearmix Dissolver(M. Technique Co., Ltd.) is desirable. The rotatory dispersing apparatusis not particularly limited as long as the whole solution can be made toflow mildly. When stirring is strong, the emulsified particles of PLGAor PLA may break down, the distribution width may become wider, and thedispersion state of the biologically active substance may collapse.

When a biologically active substance is a lipophilic biologically activesubstance, the step of forming particles can be suitably performedaccording to the above description, and a microsphere can bemanufactured. When a biologically active substance is a hydrophilicbiologically active substance, the hydrophilic biologically activesubstance is dispersed in a good solvent of PLGA or PLA using, forexample, a dispersing agent, whereby the step of forming particles canbe similarly performed to produce a microsphere.

In addition, when a biologically active substance is a hydrophilicbiologically active substance, the hydrophilic biologically activesubstance is dissolved in an aqueous solvent such as water together witha stabilizer, if necessary, and is mixed with a solution in which PLGAor PLA is dissolved in a good solvent of PLGA or PLA, to prepare a w/oemulsion; and the above step of forming particles is performed using thew/o emulsion as a solution of PLGA or PLA and the biologically activesubstance, and using the above pulverizing apparatus. For preparing thew/o emulsion, an intermittent shaking method, a propeller type stirringapparatus, a method by a mixer using a turbine type stirring apparatus,a colloid mill method, a homogenizer method, and an ultrasonicirradiation method can be used. Using the above pulverizing apparatus,this w/o emulsion of the solution of PLGA or PLA and a physiologicallyactive substance, and a solution containing a poor solvent of PLGA orPLA are continuously added to prepare emulsified particles as a w/o/wemulsion; and the good solvent is removed from the produced particles toprecipitate the microsphere of the present invention. This obtainedmicrosphere may be used as it is, but it is also possible to further addan excipient (mannitol, sorbitol, lactose, glucose, etc.), redispersethe mixture, and freeze dry or spray dry the mixture, to be solidified.A more stable sustained release injection formulation can be obtained,by adding distilled water for injection or an appropriate dispersionmedium to this solidified microsphere when used.

<Step of Filtration and Sterilization>

Sterile filtration of the prepared solution containing PLGA or PLA and abiologically active substance agent and the solution of a poor solventis preferably performed prior to the step pf forming particles, ifdesired. The solution containing a poor solvent can be sterilized byfiltration using a hydrophilic filter, and the solution of PLGA or PLAand a biologically active substance can be sterilized by filtrationusing a hydrophobic filter. A bore diameter of the filters used forfiltration is preferably 0.1 μm to 0.45 μm, more preferably 0.2 μm.

The above filter for sterile filtration is not particularly limited, andmay be appropriately selected according to the intended purpose. Forexample, for sterile filtration of the solution containing a poorsolvent, a hydrophilic filter such as polyvinylidene fluoride (PVDF) andpolyethersulfone may be used. For sterile filtration of the solution ofPLGA or PLA and a biologically active substance, a hydrophobic filtersuch as polytetrafluoroethylene (PTFE) may be used. The filter forsterile filtration is not limited to the material described here, but itis necessary to be selected depending on adsorption of the medicine anda kind of the solvent.

<Step of Removing a Good Solvent>

In the step of removing a good solvent, a good solvent is removed fromthe emulsified particles containing PLGA or PLA and a biologicallyactive substance. The step of removing a good solvent is notparticularly limited, and may be appropriately selected according to theintended purpose, as long as the good solvent can be removed from theemulsified particles in the state that a biologically active substanceis uniformly dispersed in the microsphere. The step of removing a goodsolvent includes, for example, a method of evaporating and removing thegood solvent from the fluid, by at least one of heating the fluid withstirring, flowing a gas such as nitrogen on a surface of the fluid, andreducing a pressure of the fluid. Flowing a gas such as nitrogen on asurface of the fluid is preferable. It is preferable in many cases toremove a good solvent quickly for maintaining the state that abiologically active substance is uniformly dispersed in the microsphere.It is preferable in some cases to remove a good solvent slowly. A timeof removing the good solvent may be, for example, 30 minutes to 12hours, preferably 1 to 10 hours, and more preferably 1 to 5 hours.

A temperature in removing a good solvent depends on a kind of the goodsolvent. It is necessary to perform at a suitable temperature between ahigh temperature near the boiling point of the good solvent and a lowtemperature, while observing a cross section of the microsphere. When aconcentration of PLGA or PLA is low, it is often seen that a volume ofparticles significantly changes during removing a good solvent, and thebiologically active substance dispersed in advance aggregates then.Attention should be paid to that.

<Other Steps>

Other steps include, for example, a solvent composition preparation, aclassification step, a particle cleaning step, and the like. Normally,coarse powder cut or fine powder cut is performed in the classificationstep, but the particles produced in the present invention do notsubstantially need the classification step. However, a classificationstep may be included just in case.

By the above production method, it is possible to produce a microspherehaving a particle diameter of 1 μm to 150 μm in which a biologicallyactive substance is uniformly dispersed. Namely, it is possible toproduce a microsphere wherein a variation coefficient of area ratios insix regions is 0.35 or less, wherein the area ratios in six regions arecalculated by (s/A)×100(%) wherein the six regions are prepared bypreparing a cross section observation sample obtained by cutting themicrosphere; observing the cross section observation sample with anelectron microscope at a magnification capable of confirming thebiologically active substance in the microsphere or a highermagnification; and dividing the observed image into six regions; and Ais an area of a respective divided region, and s is a sum of crosssection areas of the biologically active substance included in therespective divided region.

EXAMPLE

Hereinafter, the present invention is explained in more detail withreference to Examples, but the present invention is not limited only tothese Examples.

Reference Example 1

In Reference Example 1, microspheres (PLGA microparticles) without abiologically active substance were prepared. Using the microspheres ofReference Example 1 as an index, cross sections of the microspheres ofExamples and Comparative Examples were observed as SEM images, and thedispersion states of the biologically active substance in themicrospheres of Examples and Comparative Examples were confirmed below.

<Preparation of PLGA Solution and Aqueous PVA Solution>

Dichloromethane (Kanto Chemical Co., Inc.) was added to lacticacid-glycolic acid copolymer (Resomer RG504, Evonik AG) so that theconcentration was 13% by mass. Lactic acid-glycolic acid copolymer wasdissolved using a high-speed rotatory dispersing apparatus ClearmixDissolver (M. Technique Co., Ltd.) to obtain PLGA solution. Thereafter,the solution was filtrated with a 0.2 μm vent filter (ϕ 62, Merck KGaA).Ion exchanged water was added to polyvinyl alcohol (PVA, EG-40P, NipponSynthetic Chemical Industry Co., Ltd.) so that the concentration was1.5% by mass, and polyvinyl alcohol was dissolved using a high-speedrotatory dispersing apparatus Clearmix (M. Technique Co., Ltd.) toobtain an aqueous PVA solution. Thereafter, the solution was filtratedwith a hydrophilic PVDF membrane filter (ϕ 47, Merck KGaA). The aqueousPVA solution was added in a tank for collecting PLGA emulsifiedparticles beforehand, and was slowly stirred to an extent that thesolution surface just moved.

<Preparation of Microsphere (PLGA Microparticles)>

As the step of forming particles, the prepared PLGA solution and theaqueous PVA solution were mixed using the pulverizing apparatusdescribed in JP 2011-189348. Here, the pulverizing apparatus describedin JP 2011-189348 is an apparatus described in FIG. 25 of thepublication, in which the opening of the second part d20 has aconcentric annular shape which is surrounding the central opening of theprocessing surface 2 which is a ring-shaped disc, and a disk diameter is75 mm. Specifically, the prepared aqueous PVA solution was introducedfrom the first introduction path d1 into the space between theprocessing surfaces 1 and 2 at 0.02 MPaG, at 65 mL/min and at 30° C.,and the prepared PLGA solution was introduced from the secondintroduction path d2 into the space between the processing surfaces 1and 2 at 0.65 MPaG, at 20 mL/min and at 30° C. at the rotational speedof the processing member 10 of 2,000 rpm, and the aqueous PVA solutionand the PLGA solution were mixed in a forced thin film to prepare PLGAemulsified particles containing dichloromethane into the space betweenthe processing surfaces 1 and 2. The fluid containing PLGA emulsifiedparticles (hereinafter, PLGA emulsified particle dispersion) in thespace between the processing surfaces 1 and 2 was discharged from thespace between the processing surfaces 1 and 2 of the pulverizingapparatus. The discharged PLGA emulsified particle dispersion wascollected in a recovery tank.

Next, as the step of removing a solvent, argon gas was blown onto thefluid surface to remove dichloromethane over 3.5 hours, while stirringthe discharged fluid at a peripheral speed of 4.7 m/sec using ClearmixDissolver (M. Technique Co., Ltd.), to obtain a suspension containingPLGA microparticles (PLGA microparticle suspension). The averagevolume-based particle diameter of the obtained PLGA microparticles was34.0 μm. Representative particles were frozen with liquid nitrogen, andan FIB cross section was prepared, and an SEM image (FIG. 4) wasobserved.

As shown in FIG. 4, it was confirmed that a particle-like mass or anempty hole was not present in the FIB section. Further, it was foundthat the cross section of the microsphere of Reference Example 1 couldbe used as an index in observation of cross sections of the microspheresof Examples and Comparative examples.

Example 1

<Preparation of Solution of PLGA and Biologically Active Substance andAqueous PVA Solution>

64.5% by mass of dichloromethane (Kanto Chemical Co., Inc.) and 25% bymass of acetone (Kanto Chemical Co., Inc.) were added to lacticacid-glycolic acid copolymer (Resomer RG752H, Evonik AG) and curcumin(FUJIFILM Wako Pure Chemical Corporation, Wako special grade) as abiologically active substance, so that the concentration of lacticacid-glycolic acid copolymer was 10% by mass, and the concentration ofcurcumin was 0.5% by mass. Lactic acid-glycolic acid copolymer andcurcumin were dissolved using a high-speed rotatory dispersing apparatusClearmix Dissolver (M. Technique Co., Ltd.) to obtain a solution of PLGAand the biologically active substance. Thereafter, the solution wasfiltrated with a 0.2 μm air vent filter (ϕ 62, Merck KGaA). An aqueousPVA solution was prepared in the same manner as in Reference Example 1.The aqueous PVA solution was added in a tank for collecting emulsifiedparticles of PLGA and the biologically active substance beforehand, andwas slowly stirred to an extent that the solution surface just moved.

<Preparation of Microsphere>

As the step of forming particles, the prepared solution of PLGA and thebiologically active substance and the aqueous PVA solution were mixedusing the pulverizing apparatus described in JP 2011-189348 in the samemanner as in Reference Example 1. Specifically, the prepared aqueous PVAsolution was introduced from the first introduction path d1 into thespace between the processing surfaces 1 and 2 at 0.0 MPaG, at 50 mL/minand at 25° C., and the prepared solution of PLGA and the biologicallyactive substance was introduced from the second introduction path d2into the space between the processing surfaces 1 and 2 at 0.3 MPaG, at16 mL/min of 25° C. at the rotational speed of the processing member 10of 5,000 rpm, and the aqueous PVA solution and the solution of PLGA andthe biologically active substance were mixed in a forced thin film toprepare emulsified particles of PLGA and the biologically activesubstance containing dichloromethane in the space between the processingsurfaces 1 and 2. The fluid containing the emulsified particles of PLGAand the biologically active substance (hereinafter, emulsified particledispersion of PLGA and the biologically active substance) in the spacebetween the processing surfaces 1 and 2 was discharged from the spacebetween the processing surfaces 1 and 2 of the pulverizing apparatus.The emulsified particle dispersion of PLGA and the biologically activesubstance was collected in a recovery tank keeping the pressure of 0.03MPaG for collecting the discharged emulsified particle dispersion ofPLGA and the biologically active substance.

Next, as the step of removing a solvent, argon gas was blown on thefluid surface to remove dichloromethane and acetone over 3.5 hours,while stirring the discharged fluid at a peripheral speed of 4.7 msecusing Clearmix Dissolver (M. Technique Co., Ltd.), to obtain asuspension containing microspheres (microsphere suspension). The averagevolume-based particle diameter of the obtained microspheres was 7.5 μm.Representative particles were frozen with liquid nitrogen, and an FIBcross section was prepared, and an SEM image (FIG. 6-1) was observed. Tothe obtained particle cross section of the SEM image, was averagingprocess performed in the pixel range of 3×3 using a commercial imageanalysis software iTEM (TEM camera control, image analysis software,EMSIS GmbH); and contrast optimization was performed by a process ofhighlighting the edge part. Then, a binarization process, and a processof removing noises and highlighting particles with low contrast by imageprocessing were performed; and a second averaging process in the pixelrange of 3×3, and a process of highlighting the edge part wereperformed. FIG. 6-2 shows an image in which the particle cross sectionwas divided into six regions (Region 1 to Region 6) on the imagelatitudinally every 60° around the center point of the maximum diameteras a center.

The variation coefficient of the area ratios: (s/A)×100(%), wherein A isan area of a respective divided region, and s is a sum of cross sectionareas of the biologically active substance included in the respectivedivided region, in the FIB cross section of curcumin particles of FIG.6-2, was 0.161.

Example 2

A suspension containing microspheres was prepared in the same manner asin Example 1 except that polylactic acid (Resomer R202H, Evonik AG) wasused instead of lactic acid-glycolic acid copolymer (Resomer RG752H,Evonik AG). The average volume-based particle diameter of the obtainedmicrospheres was 7.3 μm. Representative particles were frozen withliquid nitrogen, and an FIB cross section was prepared, and an SEM imagewas observed.

The observed SEM image was image analyzed in the same manner as inExample 1. The variation coefficient of the area ratios: (s/A)×100(%),wherein A is an area of a respective divided region, and s is a sum ofcross section areas of the biologically active substance included in therespective divided region, was 0.214.

Example 3

Dichloromethane (Kanto Chemical Co., Inc.) was added to lacticacid-glycolic acid copolymer (Resomer RG504, Evonik AG) and progesterone(Sigma-Aldrich Co., LLC) as a biologically active substance, so that theconcentration of lactic acid-glycolic acid copolymer was 13% by mass,and the concentration of progesterone was 1.0% by mass. Lacticacid-glycolic acid copolymer and progesterone were dissolved using ahigh-speed rotatory dispersing apparatus Clearmix Dissolver (M.Technique Co., Ltd.) to obtain a solution of PLGA and the biologicallyactive substance. Thereafter, the solution was filtrated with a 0.2 μmair vent filter (ϕ 62, Merck KGaA). An aqueous PVA solution was preparedin the same manner as in Reference Example 1. The aqueous PVA solutionwas added in a tank for collecting emulsified particles of PLGA and thebiologically active substance beforehand, and was slowly stirred to anextent that the solution surface just moved.

<Preparation of Microsphere>

As the step of forming particles, the prepared solution of PLGA and thebiologically active substance and the aqueous PVA solution were mixedusing the pulverizing apparatus described in JP 2011-189348 in the samemanner as in Reference Example 1. Specifically, the prepared aqueous PVAsolution was introduced from the first introduction path d1 into thespace between the processing surfaces 1 and 2 at 0.0 MPaG, at 50 mL/minand at 30° C., and the prepared solution of PLGA and the biologicallyactive substance was introduced from the second introduction path d2into the space between the processing surfaces 1 and 2 at 0.35 MPaG, at16 mL/min of 30° C. at the rotational speed of the processing member 10of 1,700 rpm, and the aqueous PVA solution and the solution of PLGA andthe biologically active substance were mixed in a forced thin film toprepare emulsified particles of PLGA and the biologically activesubstance containing dichloromethane in the space between the processingsurfaces 1 and 2. The fluid containing the emulsified particles of PLGAand the biologically active substance (hereinafter, emulsified particledispersion of PLGA and the biologically active substance) in the spacebetween the processing surfaces 1 and 2 was discharged from the spacebetween the processing surfaces 1 and 2 of the pulverizing apparatus.The emulsified particle dispersion of PLGA and the biologically activesubstance was collected in a recovery tank keeping the pressure of 0.02MPaG for collecting the discharged emulsified particle dispersion ofPLGA and the biologically active substance.

The step of removing a solvent was performed in the same manner as inExamples 1 and 2. The average volume-based particle diameter of theobtained microspheres was 34.8 μm. Representative particles were frozenwith liquid nitrogen, and an FIB cross section was prepared, and an SEMimage (FIG. 7-1) was observed.

The observed SEM image was image analyzed in the same manner as inExamples 1 and 2. FIG. 7-2 shows an image prepared by enlarging the SEMimage and a binarization process The variation coefficient of the arearatios: (s/A)×100(%), wherein A is an area of a respective regionobtained by dividing into six regions on the SEM image latitudinallyevery 60° around the center point of the maximum diameter as a center,and s is a sum of cross section areas of the biologically activesubstance included in the respective divided region, was 0.056.

Example 4

Dichloromethane (Kanto Chemical Co., Inc.) was added to lacticacid-glycolic acid copolymer (Resomer RG504, Evonik AG) and probcol(FUJIFILM Wako Pure Chemical Corporation, for cell biochemistry) as abiologically active substance, so that the concentration of lacticacid-glycolic acid copolymer was 13% by mass, and the concentration ofprobcol was 1.0% by mass. Lactic acid-glycolic acid copolymer andprobcol were dissolved using a high-speed rotatory dispersing apparatusClearmix Dissolver (M. Technique Co., Ltd.) to obtain a solution of PLGAand the biologically active substance. Thereafter, the solution wasfiltrated with a 0.2 μm air vent filter (ϕ 62, Merck KGaA). An aqueousPVA solution was prepared in the same manner as in Reference Example 1.The aqueous PVA solution was added in a tank for collecting emulsifiedparticles of PLGA and the biologically active substance beforehand, andwas slowly stirred to an extent that the solution surface just moved.

The step of removing a solvent was performed in the same manner as inExamples 1 to 3. The average volume-based particle diameter of theobtained microspheres was 32.5 μm. Representative particles were frozenwith liquid nitrogen, and an FIB cross section was prepared, and an SEMimage (FIG. 8-1) was observed.

The observed SEM image was image analyzed in the same manner as inExample 4. FIG. 8-2 shows an image prepared by a binarization processThe variation coefficient of the area ratios: (s/A)×100(%), wherein A isan area of a respective region obtained by dividing into six regions onthe SEM image latitudinally every 60° around the center point of themaximum diameter as a center, and s is a sum of cross section areas ofthe biologically active substance included in the respective dividedregion, was 0.235.

Comparative Example 1

As the step of forming particles, an emulsified particle dispersion ofPLGA and the biologically active substance was prepared in the samemanner as in Examples 1 and 2. Next, as the step of removing a solvent,dichloromethane was removed in the atmosphere from the collecteddischarged fluid over 42 hours, while stirring the discharged fluid at aperipheral speed of 4.7 m/sec using Clearmix Dissolver (M. TechniqueCo., Ltd.), to obtain a suspension containing microspheres (microspheresuspension). The average volume-based particle diameter of the obtainedmicrospheres was 31.8 μm. Representative particles were frozen withliquid nitrogen, and an FIB cross section was prepared, and an SEM imagewas observed.

The observed SEM image was image analyzed in the same manner as inExamples 1 to 3. FIG. 9 shows an image prepared by a binarizationprocess The variation coefficient of the area ratios: (s/A)×100(%),wherein A is an area of a respective region obtained by dividing intosix regions on the SEM image concentrically by dividing the radius intosix equal parts from the center point of the maximum diameter, and s isa sum of cross section areas of the biologically active substanceincluded in the respective divided region, was 0.468.

Comparative Example 2

A dispersion containing microspheres was prepared with the sameformulation as in Example 4, by performing the step of forming particlesand the step of removing a solvent under the same conditions as inExample 4, except that dissolution of PLGA and the medicine wasperformed by stirring for 10 minutes with a propeller type stirringapparatus (Three-One Motor). The average volume-based particle diameterof the obtained microspheres was 29.8 μm. Representative particles werefrozen with liquid nitrogen, and an FIB cross section was prepared, andan SEM image (FIG. 10) was observed.

The observed SEM image was image analyzed in the same manner as inExamples 1 to 4. The variation coefficient of the area ratios:(s/A)×100(%), wherein A is an area of a respective region obtained bydividing into six regions on the SEM image latitudinally every 60°around the center point of the maximum diameter as a center, and s is asum of cross section areas of the biologically active substance includedin the respective divided region, was 0.357.

Comparative Example 3

69.75% by mass of dichloromethane (Kanto Chemical Co., Inc.) and 25% bymass of acetone (Kanto Chemical Co., Inc.) were added to lacticacid-glycolic acid copolymer (Resomer RG504, Evonik AG) and curcumin(FUJIFILM Wako Pure Chemical Corporation, Wako special grade) as abiologically active substance, so that the concentration of lacticacid-glycolic acid copolymer was 5.0% by mass, and the concentration ofcurcumin was 0.25% by mass. Lactic acid-glycolic acid copolymer andcurcumin were dissolved using a high-speed rotatory dispersing apparatusClearmix Dissolver (M. Technique Co., Ltd.) to obtain a solution of PLGAand the biologically active substance. Thereafter, the solution wasfiltrated with a 0.2 μm air vent filter (ϕ 62, Merck KGaA). An aqueousPVA solution was prepared in the same manner as in Reference Example 1.The aqueous PVA solution was added in a tank for collecting emulsifiedparticles of PLGA and the biologically active substance beforehand, andwas slowly stirred to an extent that the solution surface just moved.

The step of forming particles and the step of removing a solvent wereperformed to obtain a dispersion containing microspheres in the samemanner as in Example 1. The average volume-based particle diameter ofthe obtained microspheres was 6.8 μm. Representative particles werefrozen with liquid nitrogen, and an FIB cross section was prepared, andan SEM image (FIG. 11) was observed.

The observed SEM image was image analyzed in the same manner as inExamples 1 to 4. The variation coefficient of the area ratios:(s/A)×100(%), wherein A is an area of a respective region obtained bydividing into six regions on the SEM image latitudinally every 60°around the center point of the maximum diameter as a center, and s is asum of cross section areas of the biologically active substance includedin the respective divided region, was 0.386.

Comparative Example 4

69.5% by mass of dichloromethane (Kanto Chemical Co., Inc.) and 25% bymass of acetone (Kanto Chemical Co., Inc.) were added to lacticacid-glycolic acid copolymer (Resomer RG504, Evonik AG) and progesterone(Sigma-Aldrich Co., LLC) as a biologically active substance, so that theconcentration of lactic acid-glycolic acid copolymer was 5.0% by mass,and the concentration of curcumin was 0.3% by mass. Lactic acid-glycolicacid copolymer and progesterone were dissolved using a high-speedrotatory dispersing apparatus Clearmix Dissolver (M. Technique Co.,Ltd.) to obtain a solution of PLGA and the biologically activesubstance. Thereafter, the solution was filtrated with a 0.2 μm air ventfilter (ϕ 62, Merck KGaA). An aqueous PVA solution was prepared in thesame manner as in Reference Example 1. The aqueous PVA solution wasadded in a tank for collecting emulsified particles of PLGA and thebiologically active substance beforehand, and was slowly stirred to anextent that the solution surface just moved.

The step of forming particles and the step of removing a solvent wereperformed to obtain a dispersion containing microspheres in the samemanner as in Example 3. The average volume-based particle diameter ofthe obtained microspheres was 21.6 μm. Representative particles werefrozen with liquid nitrogen, and an FIB cross section was prepared, andan SEM image (FIG. 12) was observed.

The observed SEM image was image analyzed in the same manner as inExamples 1 to 4. The variation coefficient of the area ratios:(s/A)×100(%), wherein A is an area of a respective region obtained bydividing into six regions on the SEM image latitudinally every 60°around the center point of the maximum diameter as a center, and s is asum of cross section areas of the biologically active substance includedin the respective divided region, was 1.094.

Apart of the conditions of Examples 1 to 4, Comparative Examples 1 to 4,and Reference Example 1 (containing only PLGA) is shown in Table 1.

TABLE 1 Comparative Comparative Comparative Comparative ReferenceExample 1 Example 2 Example 3 Example 4 Example 1 Example 2 Example 3Example 4 Example 1 Medicine Curcumin Curcumin Progesterone ProbucolProbucol Probucol Curcumin Progesterone — Concentration of 0.5% 0.5% 1%1% 1% 1% 0.25% 0.3% — medicine by mass by mass by mass by mass by massby mass by mass by mass PLGA or PLA PLGA PLA PLGA PLGA PLGA PLGA PLGAPLGA PLGA (RG752H) (R202H) (RG504) (RG504) (RG504) (RG504) (RG504)(RG504) (RG504) Concentration of 10% 10% 13% 13% 13% 13% 5% 5% 13% PLGAor PLA by mass by mass by mass by mass by mass by mass by mass by massby mass Drying condition Argon flow Argon flow Argon flow Argon flow Inatmosphere Argon flow Argon flow Argon flow Argon flow 3.5 hours 3.5hours 3.5 horns 3.5 hours 42 hours 3.5 hours 3.5 hours 3.5 horns 3.5horns Preparation High speed High speed High speed High speed High speedPropeller High speed High speed High speed condition of stirringstirring stirring stirring stirring stirring stirring stirring solutionof PLGA or PLA and biologically acitve substance Particle 7.5 μm 7.3 μm34.8 μm 32.5 μm 31.8 μm 29.8 μm 6.8 μm 21.6 μm 34.0 μm diameter ofmicrosphere

Area ratios (%) in respective regions, standard deviations, averages andvariation coefficients (CV values) of a microsphere having arepresentative particle diameter of Examples 1 to 4, ComparativeExamples 1 to 4, and lueplin are shown in Table 2.

TABLE 2 Area ratio (%) Standard CV Region 1 Region 2 Region 3 Region 4Region 5 Region 6 deviation Average value Example 1 43.28 46.53 41.9635.68 31.60 32.38 6.20 38.57 0.161 Example 2 31.32 32.63 46.82 30.1148.15 35.66 8.00 37.45 0.214 Example 3 34.68 35.51 38.12 33.93 35.1138.99 2.02 36.06 0.056 Example 4 28.64 30.56 36.89 17.29 33.46 35.867.16 30.45 0.235 Comparative 14.28 21.68 4.62 23.56 22.13 31.56 9.1919.64 0.468 Example 1 Comparative 32.79 15.62 13.18 34.92 31.55 28.919.35 26.16 0.357 Example 2 Comparative 25.62 8.65 14.53 21.88 29.5428.23 8.25 21.41 0.386 Example 3 Comparative 2.38 3.50 23.62 21.55 3.122.12 10.26 9.38 1.094 Example 4 Leuplin 22.02 25.12 5.37 3.40 1.31 2.8010.64 10.00 1.063

As can be seen from Tables 1 and 2, in Examples 1 and 2, the variationcoefficients of area ratios of occupation of the biologically activesubstance in respective regions in the particles, were 0.35 or less, andthe biologically active substance was uniformly dispersed in theparticles, even when either of PLGA or PLA was used. In ComparativeExample 3, when the concentration of PLGA was decreased, uniformity inthe particles was lowered, and the variation coefficients of area ratiosof occupation of the biologically active substance in respectiveregions, became bigger to 0.386, even when conditions in the step offorming particles and the step of removing a solvent were the same asthose in Example 1.

In Example 4 and Comparative Example 1, the particle diameter of themicroparticles of the biologically active substance varied according todifference of the drying conditions and drying time. In ComparativeExample 1 in which the drying time was long, uniformity of thebiologically active substance dispersed in the particles was lowered,and the variation coefficient of area ratios of occupation of thebiologically active substance in respective regions, became bigger to0.468. In Comparative Examples 3 and 4 in which the concentrations ofPLGA and the medicine were decreased compared with those in Examples,percentage of contraction during drying became bigger, and thebiologically active substance was biased in the particles, and thevariation coefficients of area ratios of occupation of the biologicallyactive substance in respective regions, exceeded 0.35.

INDUSTRIAL APPLICABILITY

The present invention provides a microsphere capable of appropriatelycontrolling the initial release amount of a biologically activesubstance and its release rate during a subsequent release period, andcontinuously releasing the biologically active substance in vivo for apredetermined period of time.

1. A microsphere comprising a lactic acid-glycolic acid copolymer (PLGA)or polylactide (PLA) as a main component, in which a biologically activesubstance is uniformly dispersed, wherein an average volume-basedparticle diameter of the microsphere is 1 μm or more and 150 μm or less,and a variation coefficient of area ratios in six regions is 0.35 orless, wherein the area ratios in six regions are calculated by(s/A)×100(%) wherein the six regions are prepared by preparing a crosssection observation sample obtained by cutting the microsphere;observing the cross section observation sample with an electronmicroscope at a magnification capable of confirming the biologicallyactive substance in the microsphere or a higher magnification; anddividing the electron microscope observation image into six regions; andA is an area of a respective divided region, and s is a sum of crosssection areas of the biologically active substance included in therespective divided region.
 2. The microsphere according to claim 1,wherein the biologically active substance is a lipophilic biologicallyactive substance.
 3. The microsphere according to claim 1, wherein anaverage volume-based particle diameter of the dispersed biologicallyactive substance is 5 nm to 500 nm.
 4. The microsphere according toclaim 1, wherein a content of the biologically active substance in themicrosphere is 0.35 to 1.5% by mass.
 5. A sustained release formulationcomprising the microsphere according to claim
 1. 6. A sustained releaseformulation comprising the microsphere according to claim
 2. 7. Asustained release formulation comprising the microsphere according toclaim
 3. 8. A sustained release formulation comprising the microsphereaccording to claim 4.